1 2 An Extensive Program of Periodic Alternative Splicing Linked to Cell Cycle 3 Progression 4 5 6 Authors: 7 Daniel Dominguez1,2, Yi-Hsuan Tsai1,3, Robert Weatheritt 4, Yang Wang1,2, 8 Benjamin J. Blencowe * 4, Zefeng Wang* 1,5 9 10 Affiliations: 11 12 1 Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 13 2 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel 14 Hill, NC 15 3 Program in Bioinformatics and Computational Biology, University of North Carolina at Chapel 16 Hill, Chapel Hill, NC, 17 4 Donnelly Centre and Department of Molecular Genetics, University of Toronto, Canada 18 5 Key Lab of Computational Biology, CAS-MPG Partner Institute for Computational Biology, 19 Chinese Academy of Science, Shanghai, China 20 21 * Correspondence to: [email protected]; [email protected] 22 23 24 Abstract 25 Progression through the mitotic cell cycle requires periodic regulation of gene function at 26 the levels of transcription, translation, protein-protein interactions, post-translational 27 modification and degradation. However, the role of alternative splicing (AS) in the temporal 28 control of cell cycle is not well understood. By sequencing the human transcriptome through two 29 continuous cell cycles, we identify ~1,300 genes with cell cycle-dependent AS changes. These 30 genes are significantly enriched in functions linked to cell cycle control, yet they do not 31 significantly overlap genes subject to periodic changes in steady-state transcript levels. Many of 32 the periodically spliced genes are controlled by the SR protein kinase CLK1, whose level 33 undergoes cell cycle-dependent fluctuations via an auto-inhibitory circuit. Disruption of CLK1 34 causes pleiotropic cell cycle defects and loss of proliferation, whereas CLK1 over-expression is 35 associated with various cancers. These results thus reveal a large program of CLK1-regulated 36 periodic AS intimately associated with cell cycle control. 37 38 39 40 Impact statement 41 42 We reveal extensive periodic regulation of alternative splicing during the cell cycle in 43 genes linked to cell cycle functions, and involving an auto-inhibitory mechanism employing the 44 SR protein kinase CLK1. 2 45 Introduction 46 47 Alternative splicing (AS) is a critical step of gene regulation that greatly expands 48 proteomic diversity. Nearly all (>90%) human genes undergo AS and a substantial fraction of 49 the resulting isoforms are thought to have distinct functions (Pan et al., 2008; Wang et al., 2008). 50 AS is tightly controlled, and its mis-regulation is a common cause of human diseases (Wang and 51 Cooper, 2007). Generally, AS is regulated by cis-acting splicing regulatory elements that recruit 52 trans-acting splicing factors to promote or inhibit splicing (Matera and Wang, 2014; Wang and 53 Burge, 2008). Alterations in splicing factor expression have been observed in many cancers and 54 are thought to activate cancer-specific splicing programs that control cell cycle progression, 55 cellular proliferation and migration (David and Manley, 2010; Oltean and Bates, 2014). 56 Consistent with these findings, several splicing factors function as oncogenes or tumor 57 suppressors (Karni et al., 2007; Wang et al., 2014), and cancer-specific splicing alterations often 58 affect genes that function in cell cycle control (Tsai et al., 2015). 59 Progression through the mitotic cell cycle requires periodic regulation of gene function 60 that is primarily achieved through coordination of protein levels with specific cell cycle stages 61 (Harashima et al., 2013; Vermeulen et al., 2003). This temporal coordination enables timely 62 control of molecular events that ensure accurate chromatin duplication and daughter cell 63 segregation. Periodic gene function is conventionally thought to be achieved through stage- 64 dependent gene transcription (Bertoli et al., 2013), translation (Grabek et al., 2015), protein- 65 protein interactions (Satyanarayana and Kaldis, 2009), post-translational protein modifications, 66 and ubiquitin-dependent protein degradation (Mocciaro and Rape, 2012). Although AS is one of 67 the most widespread mechanisms involved in gene regulation, the relationship between the 68 global coordination of AS and the cell cycle has not been investigated. 3 69 Major families of splicing factors include the Serine-Arginine rich proteins (SR) proteins 70 and the heterogeneous nuclear ribonucleoproteins (hnRNPs), whose levels and activities vary 71 across cell types. SR proteins generally contain one or two RNA recognition motifs (RRMs) and 72 a domain rich in alternating Arg and Ser residues (RS domain). Generally, RRM domains confer 73 RNA binding specificity while the RS domain mediates protein-protein and protein-RNA 74 interactions to affect splicing (Long and Caceres, 2009; Zhou and Fu, 2013). Post-translational 75 modifications of SR proteins, most notably phosphorylation, modulate their splicing regulatory 76 capacity by altering protein localization, stability or activity (Gui et al., 1994; Lai et al., 2003; 77 Prasad et al., 1999; Shin and Manley, 2002). Dynamic changes in SR protein phosphorylation 78 have been detected after DNA damage (Edmond et al., 2011; Leva et al., 2012) and during the 79 cell cycle (Gui et al., 1994; Shin and Manley, 2002), suggesting that regulation of AS may have 80 important roles in cell cycle control. However, the functional consequences of SR protein 81 (de)phosphorylation during the cell cycle are largely unclear. 82 Through a global-scale analysis of the human transcriptome at single-nucleotide 83 resolution through two continuous cell cycles, we have identified widespread periodic changes in 84 AS that are coordinated with specific stages of the cell cycle. These periodic AS events belong to 85 a set of genes that is largely separate from the set of genes periodically regulated during the cell 86 cycle at the transcript level, yet the AS regulated set is significantly enriched in cell cycle- 87 associated functions. We further demonstrate that a significant fraction of the periodic AS events 88 is regulated by the SR protein kinase, CLK1, and that CLK itself is also subject to cell cycle- 89 dependent regulation. Moreover, inhibition or depletion of CLK1 causes pleiotropic defects in 90 mitosis that lead to cell death or G1/S arrest, suggesting that the temporal regulation of splicing 91 by CLK1 is critical for cell cycle progression. The discovery of periodic AS thus reveals a 4 92 widespread yet previously underappreciated mechanism for the regulation of gene function 93 during the cell cycle. 94 95 Results 96 Alternative splicing is coordinated with different cell cycle phases 97 To systematically investigate the regulation of AS during the cell cycle, we performed an 98 RNA-Seq analysis of synchronously dividing cells using a total of 2.3 billion reads generated 99 across all stages (G1, S, G2 and M) of two complete rounds of the cell cycle (Figure 1-figure 100 supplement 1A). To maximize the detection of regulated AS events, we used the complementary 101 analysis pipelines, MISO and VAST-TOOLS (Katz et al., 2010; Irimia et al., 2014). These 102 pipelines have different detection specificities and employ partially overlapping reference sets of 103 annotated AS events, and therefore afford a more comprehensive analysis when employed 104 together. Both pipelines were used to determine PSI (the percent of transcript with an exon 105 spliced in) and PIR (the percent of transcripts with an intron retained). Alternative exons 106 detected by both pipelines had highly correlated PSI values (Figure 1-figure supplement 1G; see 107 below). Consistent with previous results (Bar-Joseph et al., 2008; Whitfield et al., 2002), 108 transcripts from approximately 14.2% (1,182) of expressed genes displayed periodic differences 109 in steady-state levels between two or more cell cycle stages (see below). Remarkably, 15.6% 110 (1,293) of expressed genes also contained 1,747 periodically-regulated AS events, among a total 111 of ~40,000 detected splicing events (FDR < 2.5%; Figure 1A and Figure 1-figure supplement 112 1B, 1C, 1D). 113 Importantly, as has been observed previously for AS regulatory networks (Pan et al., 114 2004), the majority of genes with periodic AS events did not overlap those with periodic steady- 5 115 state changes in mRNA expression. This indicates that genes with periodic changes in AS and 116 transcript levels are largely independently regulated during the cell cycle (Figure 1B). Further 117 supporting this conclusion, we did not observe a significant correlation (positive or negative) 118 between exon PSI values and mRNA expression levels for genes with both periodic expression 119 and periodic exon skipping (data not shown). A gene ontology (GO) analysis reveals that genes 120 with periodic AS, like those with periodic transcript level changes (Bar-Joseph et al., 2008; 121 Whitfield et al., 2002), are significantly enriched in cell cycle-related functional categories, 122 including M-phase, nuclear division and DNA metabolic process (Figure 1C; adjusted P <0.05 123 for all listed categories, FDR <10%) (Supplementary Table1). Similar GO enrichment results 124 were observed when removing the relatively small fraction (10%) of periodically spliced genes 125 that also display significant mRNA expression changes across the cell cycle (Figure 1C). These 126 results thus reveal that numerous genes not previously linked to the cell cycle, as well as 127 previously defined cell cycle-associated genes thought to be constantly expressed across the cell 128 cycle, are in fact subject to periodic regulation at the level of AS (Supplementary File 1 for a full 129 list). 130 Among the different classes of AS analyzed (cassette exons, alternative 5´/3´ splice sites 131 and intron retention [IR]), periodically regulated IR events were over-represented (relative to the 132 background frequency of annotated IR events) by ~2.2 fold whereas periodically regulated 133 cassette exons, represent the next most frequent periodic class of AS (P = 2.2×10-16, Fisher’s 134 exact test, Figure 1-figure supplement 1E).